High purity 2-hydroxyphenazine and methods of making the same

Abstract

The application discloses a high-purity 2-hydroxyphenazine compound and a purification method thereof, successfully separates and purifies a large amount of 2-hydroxyphenazine compound from a green needle pseudomonas fermentation liquor, and comprises the following steps: centrifuging the green needle pseudomonas fermentation liquor to remove bacterial bodies to obtain a sample liquid; using a macroporous adsorption resin column to perform dynamic adsorption on the sample liquid until saturation adsorption; using a low-concentration ethanol aqueous solution to elute impurities on the macroporous adsorption resin column; using a higher-concentration ethanol aqueous solution as an eluent to desorb, to obtain a desorption liquid; removing the eluent in the desorption liquid to obtain 2-hydroxyphenazine purified products. The recovery rate of the method reaches 93.01+5.95%, has the advantages of simple operation, environmental protection and no pollution, and has wide application value in industrialized large-scale production.

Description

Technical Field

[0001] This invention relates to the field of technology, specifically to a high-purity 2-hydroxyphenazine and its preparation method. Background Technology

[0002] 2-Hydroxyphenazine exhibits good antagonistic effects against wheat take-all pathogens, sclerotinia pathogens, and anthracnose pathogens, showing promising application prospects. Furthermore, some progress has been made in the biosynthesis of 2-hydroxyphenazine, which can be naturally synthesized in *Pseudomonas aeruginosa*. Currently, the traditional method for separating phenazine compounds involves liquid-phase extraction using organic solvents such as ethyl acetate and chloroform. However, this method suffers from drawbacks such as complex separation processes, long processing times, and suitability only for small-scale preparations, making it unsuitable for large-scale industrial production.

[0003] Currently, the traditional separation method for obtaining 2-hydroxyphenazine mainly utilizes liquid-phase extraction with organic solvents such as ethyl acetate and chloroform. However, this traditional method has many serious drawbacks. From a process perspective, the separation process is extremely complex, involving multiple steps and delicate operations, requiring operators with high levels of professional skills and extensive experience. In terms of time cost, the entire extraction process is time-consuming, often requiring a significant amount of time from sample pretreatment to final product acquisition. Furthermore, this method is only suitable for small-scale preparations and cannot meet the needs of large-scale industrial production.

[0004] With the increasing demands of industrial production, the limitations of traditional liquid phase extraction methods are becoming increasingly apparent. The extensive use of organic solvents not only significantly increases production costs but also generates substantial amounts of organic solvent waste. The treatment of this waste is difficult and costly; improper handling can cause serious environmental pollution, contradicting modern green production principles. Furthermore, the volatilization of organic solvents poses a threat to the health of operators, necessitating specialized protective equipment and facilities.

[0005] Macroporous adsorption resins, as high-molecular polymers that generate adsorption through van der Waals forces and hydrogen bonding, have been widely used in the pharmaceutical and chemical industries for separating polycyclic and heterocyclic compounds. Their unique physicochemical properties hold promise for providing a new solution for the separation and purification of 2-hydroxyphenazine, overcoming many drawbacks of traditional organic solvent extraction methods and meeting the urgent market demand for large-scale, efficient, and environmentally friendly production of 2-hydroxyphenazine. However, there are still many technological gaps and challenges in applying macroporous adsorption resins to the separation and purification of 2-hydroxyphenazine, requiring further research and exploration. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by providing a high-purity 2-hydroxyphenazine and its preparation method.

[0007] The technical solution provided by this invention is as follows:

[0008] S1. Centrifuge the Pseudomonas aeruginosa fermentation broth and collect the supernatant as the loading solution;

[0009] S2. The sample solution was dynamically adsorbed to saturation using a macroporous adsorption resin column to capture the target compound 2-hydroxyphenazine.

[0010] S3. Use a low-concentration alcohol solution to rinse the impurities on the macroporous adsorption resin column;

[0011] S4. Use a high-concentration alcohol solution as an eluent to desorb, and obtain the eluent;

[0012] S5. Remove the eluent from the desorption solution, dry, and obtain the purified 2-hydroxyphenazine.

[0013] The macroporous adsorption resin includes HZ-818 and / or HZ-801.

[0014] Preferably, the macroporous adsorption resin includes HZ-818.

[0015] The height-to-diameter ratio of the macroporous adsorption resin column is 80-100:1; the specific surface area of ​​the macroporous adsorption resin is 800-1200 m². 2 / g, with a particle size of 0.32~1.20mm.

[0016] The macroporous adsorption resin HZ-818 is a milky white, semi-transparent spherical particle with a moisture content of 60-70 wt% and a wet true density of 1.00-1.10 g / mL.

[0017] Before packing the macroporous adsorption resin column, a pretreatment process is performed, which includes the following steps:

[0018] A. The macroporous adsorption resin is soaked in ethanol for 20-24 hours and washed with water to obtain alcohol-treated resin, wherein the concentration of ethanol is 95-98%, the washing rate is 1-2 BV / h, and the amount of water used is 2-5 BV.

[0019] B. The alcohol-treated resin is sequentially soaked in an acid solution and washed with water to obtain an acid-treated resin;

[0020] C. The acid-treated resin is successively soaked in alkaline solution and washed with water;

[0021] D. The alkali-treated resin is ultrasonically treated and then dried.

[0022] In step B, the acid solution is 0.4-0.8M HCl, and the soaking time is 4-6 hours; the water washing is performed with distilled water at a rate of 1-2 BV / h until neutral, and the amount of water used is 2-3 BV.

[0023] And / or, in step C, the alkaline solution is a 0.5-1M NaOH solution, and the soaking time is 4-6 hours; the water washing is performed with distilled water at a rate of 1-2 BV / h until neutral, and the amount of water used is 2-3 BV.

[0024] And / or, in step D, the alkali-treated resin is placed in 8-10 BV distilled water for ultrasonic treatment, with an ultrasonic frequency of 25-50 kHz, a power of 100-162.5 W, and an ultrasonic duration of 20-30 min.

[0025] Macroporous adsorption resin packed columns use a wet packing process. Specific steps are as follows:

[0026] The pretreated resin is soaked in a 95-98% ethanol aqueous solution for 20-24 hours, stirred evenly, and then poured into a chromatography column containing 0.3-0.4 BV of 95-98% ethanol solution. At the same time, the solution is released and the column wall is tapped with a rubber wash ball to avoid uneven resin packing. When the liquid level drops to 1-2 cm from the surface of the resin column, it is washed with distilled water to complete the wet packing of the resin column.

[0027] In step S2, during dynamic adsorption, the loading rate of the sample solution is 0.8-2 BV / h.

[0028] The low-concentration alcohol solution mentioned in step S3 is a low-concentration ethanol solution with a volume fraction of 40-50%, the rinsing flow rate is 0.4-0.6 BV / h, and the amount of the low-concentration ethanol solution used is 1-1.2 BV.

[0029] In step S4, the eluent is a high-concentration ethanol solution with a volume fraction of 75-95%; the desorption rate of the eluent is 0.6-1.5 BV / h, and the amount of the eluent used is 4-5 BV.

[0030] In step S2, adsorption is carried out at a temperature of 16-20℃.

[0031] In step S5, the drying step is as follows: transfer the desorbed liquid to a rotary evaporator, seal and reduce the pressure to 400-500 mmHg; heat to 80-85°C until the volume is concentrated to 6-8% of the original desorbed liquid.

[0032] This invention pretreats the resin, including ethanol soaking and water washing, followed by acid and alkali solution soaking and water washing, ultrasonic treatment, and drying, with each step working in tandem. Soaking in 95-98% ethanol for 20-24 hours removes impurities and causes swelling in the resin. Acid and alkali treatment further purifies the resin and adjusts its surface chemical properties. Ultrasonic treatment (frequency 25-50 kHz, power 100-162.5 W, duration 20-30 min) removes fine impurities from within the resin and makes its structure more uniform and stable, enhancing its adsorption performance and lifespan, ensuring stable operation throughout the entire preparation process.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. Although macroporous adsorption resins have a certain adsorption capacity for 2-hydroxyphenazine, some structurally similar compounds in the fermentation broth often compete with 2-hydroxyphenazine for adsorption sites, affecting the selectivity of the resin. Therefore, this invention conducts a comparative study on various macroporous adsorption resins. The macroporous adsorption resins HZ-818 and / or HZ-801 have better effects. Preferably, macroporous adsorption resin HZ-818 is used as the adsorbent, and a large amount of 2-hydroxyphenazine compounds are successfully separated and purified from the fermentation broth of Pseudomonas aeruginosa. The maximum adsorption capacity of HZ-818 resin for 2-hydroxyphenazine is as high as 6.8 mg / g, and it has excellent selective adsorption performance and stable physicochemical properties.

[0035] 2. Although the macroporous adsorption resin HZ-818 has a certain adsorption capacity for 2-hydroxyphenazine, in practical applications, various factors prevent the adsorption capacity from reaching the ideal value. For example, the pH value, temperature, and ionic strength of the solution may affect the adsorption performance of the resin. This invention further optimizes the process conditions, such as temperature, sample loading flow rate, and eluent concentration, achieving a recovery rate of 93.01±5.95%. The purification method of this invention has the advantages of simple operation and environmental friendliness, and has broad application value in large-scale industrial production.

[0036] 3. The sample loading rate was controlled at 0.8-2 BV / h, ensuring sufficient contact and adsorption of the target compound with the resin while avoiding incomplete adsorption due to excessively high flow rates. Eluting with a low-concentration ethanol solution (40-50% v / h) at a flow rate of 0.4-0.6 BV / h and a dosage of 1-1.2 BV effectively removed impurities from the resin column while minimizing the loss of the target compound. Desorption was performed using a high-concentration ethanol solution (75-85% v / h) at a rate of 0.6-1.5 BV / h and a dosage of 4-5 BV, ensuring efficient desorption of the target compound. Combined with an operating temperature of 16-20℃, a stable and suitable reaction environment was provided for the entire process, allowing each step to proceed efficiently and effectively, ultimately achieving a recovery rate as high as 93.01±5.95%.

[0037] 4. Compared to traditional organic solvent extraction and separation methods, this invention avoids the generation of large amounts of organic solvent waste liquid, making it more environmentally friendly. The entire preparation method has a clear operation flow, with well-defined and easily controllable conditions for each step, eliminating the need for complex and expensive equipment and highly skilled operators. For example, the desorbed liquid is dried using a rotary evaporator under specific conditions (sealed, reduced pressure to 400-500 mmHg, and concentrated by heating at 80-85°C). The operation is simple and efficient, offering significant advantages for large-scale industrial production, effectively reducing production costs and improving production efficiency and economic benefits.

[0038] 5. This invention uses a high concentration of ethanol as the eluent for desorption. Compared to methanol, ethanol is less polar, but provides a higher recovery rate for the same volume, further improving purification efficiency and economic benefits. This invention uses a high concentration of ethanol as the eluent because the solubility of 2-hydroxyphenazine in aqueous solution under neutral conditions is less than 10 mg / L. Therefore, based on the principle of "like dissolves like," this invention uses an ethanol solution with a weaker polarity than methanol for elution. Compared to methanol, using a high concentration of ethanol as the eluent provides a higher recovery rate for the same volume. Attached Figure Description

[0039] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0040] Figure 1 To determine the adsorption equilibrium of 2-hydroxyphenazine on the resin at different temperatures;

[0041] Figure 2 The desorption of 2-hydroxyphenazine on the resin by ethanol solutions of different volume fractions is shown.

[0042] Figure 3 The residual status of 2-hydroxyphenazine in the effluent of the sample solution at different flow rates;

[0043] Figure 4 A comparison chart of the desorption effects of methanol and ethanol;

[0044] Figure 5 This is a comparison of the soaking times of HCl and NaOH in Example 6;

[0045] Figure 6 This is a comparison chart showing the resin undergoing ultrasonic treatment before and after the pretreatment process. Detailed Implementation

[0046] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0047] In this invention, the HZ-818 and / or HZ-801 used have a specific surface area of ​​800-1200 m². 2 / g, with a particle size of 0.32~1.20mm.

[0048] Example 1: Determination of the Optimal Resin

[0049] (1) Use seven macroporous adsorption resins: D001, D155, HP-20, HZD-2, HZ-801, HZ-818, and HZ-016. Take three portions of each resin, weigh each portion accurately (0.5g), and soak them overnight in 1mL of 95% ethanol.

[0050] (2) Filtration and separation: wash the resin with deionized water to remove ethanol, and pour them into 250mL shake flasks respectively.

[0051] (3) Each resin was soaked and mixed with 25 mL of 30 mg / L 2-hydroxyphenazine aqueous solution, the flask was sealed with sealing film, placed in a shaker, and shaken at 28°C and 200 rpm for 24 h. The change in 2-hydroxyphenazine concentration was then detected by sampling.

[0052] (4) Filter the resin and 2-hydroxyphenazine aqueous solution to separate them, and wash the resin with deionized water 1-2 times to remove the residual 2-hydroxyphenazine solution.

[0053] (5) Each resin was soaked and mixed with 25 mL of 95% ethanol solution, the flask was sealed with sealing film, placed in a shaker, and shaken at 28°C and 200 rpm for 24 h. The concentration change of 2-hydroxyphenazine was detected by sampling. The detection results are shown in Table 1.

[0054] Table 1 Separation effect of different macroporous resins

[0055] Macroporous resin types Manufacturer Final concentration of 2-hydroxyphenazine (mg / L) D001 Zhejiang Zhengguang Industrial Co., Ltd. 0.53 D155 Shanghai Nankai Resin Co., Ltd. 1.07 HZD-2 Shanghai Huazhen Technology Co., Ltd. 0.23 HZ-801 Zhengzhou Aino Chemical Technology Co., Ltd. 13.47 HZ-818 Shanghai Huazhen Technology Co., Ltd. 19.67 HP-20 Beijing Solarbio Technology Co., Ltd. 5.21 HZ-016 Greenlink Chemical Technology Co., Ltd. 0.17

[0056] As shown in Table 1, macroporous resin HZ-818 exhibits the best separation performance. Macroporous resin HZ-818 will be selected for further research.

[0057] Example 2: Determination of sample loading solution concentration and purification temperature

[0058] (1) Accurately weigh several portions of HZ-818 macroporous resin, each portion being 1.0 g, and soak them overnight in 2 mL of 95% ethanol.

[0059] (2) Filtration and separation: wash the resin with deionized water to remove ethanol, and pour them into 250mL shake flasks respectively.

[0060] (3) Each part of resin was mixed with different concentrations of aqueous solution prepared from pure 2-hydroxyphenazine, and the shake flask was sealed with sealing film; the concentrations of 2-hydroxyphenazine were 17.5 mg / L, 40 mg / L, 80 mg / L, 140 mg / L and 240 mg / L (as shown in Table 2).

[0061] Table 2

[0062] test group number 2-Hydroxyphenazine concentration 2-1 17.5 mg / L 2-2 40mg / L 2-3 80mg / L 2-4 140mg / L 2-5 240mg / L

[0063] (4) Place the mixture of each test group into a shaker at 16℃, 28℃ and 45℃ respectively, shake at 200rpm for 24h, take samples to detect the change in 2-hydroxyphenazine concentration, calculate the change in the amount of resin adsorbed per unit mass, and draw adsorption isotherms generated by initial solutions of different concentrations at different temperatures.

[0064] Adsorption isotherms generated by initial solutions of different concentrations at different temperatures, such as Figure 1 As shown, Figure 1 The data on the horizontal axis represents the equilibrium concentration of 2-hydroxyphenazine in the solution after resin adsorption.

[0065] Depend on Figure 1 It can be seen that, under different sample solution concentrations, the adsorption effect of HZ-818 resin on 2-hydroxyphenazine gradually increases with decreasing temperature. Among 16℃, 28℃, and 45℃, the adsorption effect of HZ-818 resin is best at 16℃. After fitting the isotherm curves using the Langmuir thermodynamic equation, it was found that the maximum adsorption capacity of HZ-818 resin is 6.8 mg / g. Therefore, the maximum concentration of the sample solution is...

[0066]

[0067] In the formula, ρ Resin The wet true density of HZ-818 resin (1.00~1.10g / mL); V resin q represents the volume of resin involved in adsorption (mL); m The maximum adsorption capacity of HZ-818 for 2-hydroxyphenazine is 6.8 mg / g; V A The volume of the sample (L) is the volume of the sample.

[0068] Example 3: Determination of eluent concentration

[0069] (1) Accurately weigh several portions of HZ-818 macroporous resin, each 0.5g, and soak them overnight in 1mL of 95% ethanol to ensure that the resin is fully impregnated.

[0070] (2) Filtration and separation: wash the resin with deionized water to remove ethanol, and pour them into 250mL shake flasks respectively.

[0071] (3) Each resin sample was soaked and mixed with 25 mL of 2-hydroxyphenazine aqueous solution, the shake flask was sealed with sealing film, placed in a shaker, and shaken at 28°C and 200 rpm for 24 h. The change in 2-hydroxyphenazine concentration was measured to evaluate the adsorption effect of the resin.

[0072] (4) Washing: Filter the resin and 2-hydroxyphenazine aqueous solution to separate them. Wash the resin with deionized water 1-2 times to remove the residual 2-hydroxyphenazine solution.

[0073] (5) Elution: 25 mL of ethanol solutions with volume fractions of 95%, 85%, 75%, 65%, 55%, 45%, 35%, and 25% were added to the resin, respectively. The mixture was shaken at 200 rpm for 24 hours at 28°C. Samples were taken for analysis to evaluate the elution effect of different ethanol concentrations. The results are as follows: Figure 2 As shown;

[0074] Depend on Figure 2 It can be seen that the desorption effect of the eluent is best when the eluent is a 75-95% ethanol solution. When the eluent is a 75% ethanol solution, the desorption rate reaches 84.79±1.49%.

[0075] Example 4: Determination of Sample Loading Rate

[0076] Test strain: Pseudomonas aeruginosa GP72 (CGMCC 1748CN 1932006A);

[0077] KB plate culture medium formula: 20 g / L peptone, 15 mL / L glycerol, 0.514 g / L dipotassium hydrogen phosphate, 0.732 g / L magnesium sulfate, 20 g / L agar;

[0078] KB liquid culture medium formula: 20 g / L peptone, 15 mL / L glycerol, 0.514 g / L dipotassium hydrogen phosphate, 0.732 g / L magnesium sulfate.

[0079] The HZ-818 macroporous adsorption resin used in this embodiment is a milky white, semi-transparent spherical particle. The preferred height-to-diameter ratio of the macroporous adsorption resin column is 100:1, and the specific surface area of ​​the macroporous resin is 900 m². 2 / g, particle size 1.20mm, moisture content 60-70wt%, wet true density 1.00-1.10g / mL.

[0080] First, HZ-818 macroporous resin was soaked in 95% ethanol for 24 hours, and then washed with distilled water at a rate of 1 BV / h, using 2 BV of ethanol. Next, it was soaked in 0.8M HCl solution for 6 hours, and then washed with distilled water at a rate of 2 BV / h until neutral, using 2 BV of HCl. Finally, it was soaked in 1M NaOH solution for 6 hours, and then washed with distilled water at a rate of 2 BV / h until neutral, using 2 BV of NaOH. The alkali-treated resin was then subjected to ultrasonic treatment in 8 BV distilled water at a frequency of 25 kHz, a power of 162.5 W, and a duration of 0.5 hours, followed by drying.

[0081] Soak the resin in 95% ethanol aqueous solution for 24 hours, stir well, and then pour it into a chromatography column containing 0.3 BV of 95% ethanol aqueous solution. At the same time, open the chromatography column knob to release the solution and tap the column wall with a rubber bulb to avoid uneven resin packing. When the liquid level drops to 1-2 cm from the surface of the resin column, wash with distilled water to complete the wet packing of the resin column.

[0082] (1) Preparation of fermentation broth

[0083] The tested strain, *Pseudomonas aeruginosa* GP72, was inoculated onto KB agar plates for activation once (28°C, 12h). A single colony from the plate was picked and cultured overnight in 5 mL of KB liquid medium (28°C, 24h). The culture was then inoculated at a concentration of 1 / 1000 and fermented for 4 days.

[0084] Centrifuge the Pseudomonas aeruginosa fermentation broth at 10,000 rpm for 10 min to remove the bacterial cells, and retain the supernatant as the loading solution.

[0085] (2) The supernatant of the 6 BV fermentation broth was passed through a chromatography column packed with HZ-818 macroporous resin at flow rates of 0.96 BV / h, 1.92 BV / h, and 6 BV / h, respectively. The eluent was collected, and the concentration change was measured at 1 BV intervals. The results are as follows: Figure 3 As shown.

[0086] Depend on Figure 3 It can be seen that when the loading flow rate is 0.96–1.92 BV / h, HZ-818 resin can completely adsorb 2-hydroxyphenazine in the fermentation broth. However, when the loading flow rate is 1.92 BV / h, 2-hydroxyphenazine residue appears in the effluent when the loading volume increases to 4 BV. At a loading flow rate of 0.96 BV / h, the HZ-818 resin column can ensure complete adsorption of 2-hydroxyphenazine in 6 BV of fermentation broth.

[0087] Example 52: Purification of 2-hydroxyphenazine

[0088] 1. Treatment of Pseudomonas aeruginosa LX24 fermentation broth

[0089] The fermentation broth required for this experiment was obtained from *Pseudomonas aeruginosa* GP72 (CGMCC 1748CN 1932006A). The culture was activated once on KB agar plates (28℃, 12h). A single colony was picked from the plate and cultured overnight in 5mL of KB liquid medium. The culture was then inoculated with the culture at a concentration of 1 / 1000 for fermentation.

[0090] 2. High-speed centrifugation treatment of fermentation broth

[0091] Centrifuge the Pseudomonas aeruginosa fermentation broth at 10,000 rpm for 10 min to remove the bacterial cells, and retain the supernatant as the loading solution.

[0092] 3. Purification of 2-hydroxyphenazine compounds in fermentation broth using macroporous resin

[0093] 3.1 Resin Column Preparation

[0094] The HZ-818 macroporous adsorption resin used in this embodiment is a milky white, semi-transparent spherical particle. The preferred height-to-diameter ratio of the macroporous adsorption resin column is 100:1, and the specific surface area of ​​the macroporous resin is 900 m². 2 / g, particle size 1.20mm, moisture content 60-70wt%, wet true density 1.00-1.10g / mL.

[0095] Resin pretreatment: First, soak the HZ-818 macroporous resin in 95% ethanol for 24 hours, then wash the resin with distilled water at a rate of 1 BV / h, using 2 BV. Next, soak the resin in 0.8M HCl solution for 6 hours, washing with distilled water at a rate of 2 BV / h until neutral, using 2 BV. Finally, soak the resin in 1M NaOH solution for 6 hours, washing with distilled water at a rate of 2 BV / h until neutral, using 2 BV. Then, place the alkali-treated resin in 8 BV distilled water for ultrasonic treatment at a frequency of 25 kHz, a power of 162.5 W, and a duration of 0.5 hours, followed by drying.

[0096] Wet packing of resin column: Soak the pretreated resin in 95% ethanol aqueous solution for 24 hours, stir well, and then pour it into the chromatography column containing 0.3 BV of 95% ethanol aqueous solution. At the same time, open the chromatography column knob to release the solution and tap the column wall with a rubber washer to avoid uneven resin packing. When the liquid level drops to 1-2 cm from the surface of the resin column, wash with distilled water to complete the wet packing of resin column.

[0097] 3.2 Dynamic Adsorption: The supernatant of the obtained fermentation broth was subjected to dynamic adsorption on a macroporous adsorption resin column (purification environment temperature 16℃). The loading rate of the supernatant was 0.96 BV / h. The adsorption was allowed to reach saturation (when the concentration of 2-hydroxyphenazine in the elution solution was 4% of the initial sample solution and the total volume of the elution solution was equal to the dynamic processing capacity of the resin, the resin was defined as being in adsorption equilibrium).

[0098] 3.3 Washing: Impurities on the macroporous adsorption resin column were washed with a 40% ethanol aqueous solution at a rate of 0.6 BV / h and a volume of 1 BV of 40% ethanol aqueous solution.

[0099] 3.4 Desorption: Dynamic desorption was performed using a 75% ethanol aqueous solution to obtain the desorption solution. The desorption rate was 0.6 BV / h, and the amount of 75% ethanol aqueous solution used was 5 BV.

[0100] 3.5 Concentration and Drying: Finally, the collected eluent was transferred to a rotary evaporator, sealed and depressurized to 400 mmHg, heated to 81°C in a water bath, and the flask was rotated at 60 rpm. The volume was concentrated to 6.67% of the original eluent. The concentrated fermentation broth was then dried in a fume hood, and the residual solids were collected to obtain pure 2-hydroxyphenazine. After HPLC analysis, the purity reached up to 94.8%.

[0101] Example 6

[0102] This embodiment compares and studies the soaking time of HCl and NaOH in the resin pretreatment in step 3.1 of Example 5. The remaining steps are the same as in Example 5.

[0103] The results are as follows Figure 5 As shown, the recovery rate significantly increases with increasing treatment time and eventually converges to a uniform level. Therefore, the pretreatment method of acid soaking for 4 hours and alkali soaking for 6 hours is optimal.

[0104] Comparative Example 1

[0105] The difference between this comparative example and Example 5 is that in step 3.4, desorption is performed using a 75% methanol aqueous solution.

[0106] Ultimately, the recovery rate of the 75% methanol aqueous solution was significantly lower than that obtained by desorption using a 75% ethanol aqueous solution in Example 5, as shown in the results. Figure 4 As shown.

[0107] Comparative Example 2

[0108] The difference between this comparative example and Example 5 is that the resin is not subjected to ultrasonic treatment during the pretreatment process, and the result is as follows: Figure 6As shown, the resin treated with ultrasound exhibited a faster adsorption rate and an adsorption capacity increased by 22.6%.

[0109] In summary, this invention discloses a method for purifying 2-hydroxyphenazine compounds using macroporous adsorption resin, successfully isolating and purifying a large amount of 2-hydroxyphenazine compounds from the fermentation broth of *Pseudomonas aeruginosa*. The method includes the following steps: centrifuging the *Pseudomonas aeruginosa* fermentation broth to remove bacterial cells, obtaining a loading solution; dynamically adsorbing the loading solution onto a macroporous adsorption resin column until saturation; eluting impurities on the macroporous adsorption resin column using a low-concentration ethanol-water solution; desorbing using a higher-concentration ethanol-water solution as the eluent, obtaining an eluent; and removing the eluent from the eluent to obtain the purified 2-hydroxyphenazine. The method of this invention achieves a recovery rate of 93.01±5.95%, and has the advantages of simple operation and environmental friendliness, making it highly valuable for large-scale industrial production.

[0110] It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in these embodiments can be arbitrarily combined with each other.

Claims

1. A method for preparing high-purity 2-hydroxyphenazine, characterized in that, Includes the following steps: S1. Centrifuge the Pseudomonas aeruginosa fermentation broth and collect the supernatant as the loading solution; S2. The sample solution was dynamically adsorbed to saturation using a macroporous adsorption resin column to capture the target compound 2-hydroxyphenazine. S3. Use a low-concentration alcohol solution to rinse the impurities on the macroporous adsorption resin column; S4. Use a high-concentration alcohol solution as an eluent to desorb, and obtain the eluent; S5. Remove the eluent from the desorption solution, dry, and obtain the purified 2-hydroxyphenazine. The macroporous adsorption resin is HZ-818 and / or HZ-801; In step S2, during dynamic adsorption, the loading rate of the sample solution is 0.8-2 BV / h; adsorption is carried out at a temperature of 16-20 ℃. In step S4, the eluent is a high-concentration ethanol solution with a volume fraction of 75-95%; the desorption rate of the eluent is 0.6-1.5 BV / h, and the amount of eluent used is 4-5 BV. Before packing macroporous adsorption resin into a column, a pretreatment process is performed, which includes the following steps: A. The macroporous adsorption resin is sequentially soaked in ethanol for 20-24 h and washed with water to obtain alcohol-treated resin, wherein the concentration of ethanol is 95-98%, the washing rate is 1-2 BV / h, and the amount of water used is 2-5 BV. B. The alcohol-treated resin is sequentially soaked in an acid solution and washed with water to obtain an acid-treated resin; C. The acid-treated resin is sequentially soaked in alkaline solution and washed with water; D. The alkali-treated resin is ultrasonically treated and then dried.

2. The preparation method according to claim 1, characterized in that, In step B, the acid solution is 0.4-0.8 M HCl, and the soaking time is 4-6 h; the water washing is performed with distilled water at a rate of 1-2 BV / h until neutral, and the amount used is 2-3 BV. And / or, in step C, the alkaline solution is a 0.5-1 M NaOH solution, and the soaking time is 4-6 h; the water washing is performed with distilled water at a rate of 1-2 BV / h until neutral, and the amount of water used is 2-3 BV. And / or, in step D, the alkali-treated resin is placed in 8-10 BV water for ultrasonic treatment, with an ultrasonic frequency of 25-50 kHz, a power of 100-162.5 W, and an ultrasonic duration of 20-30 min.

3. The preparation method according to claim 1, characterized in that, The low-concentration alcohol solution mentioned in step S3 is a low-concentration ethanol solution with a volume fraction of 40-50%, the rinsing flow rate is 0.4-0.6 BV / h, and the amount of the low-concentration ethanol solution used is 1-1.2 BV.

4. The preparation method according to claim 1, characterized in that, In step S5, the drying step is as follows: transfer the desorbed liquid to a rotary evaporator, seal and reduce the pressure to 400-500 mmHg; heat to 80-85 °C until the volume is concentrated to 6-8% of the original desorbed liquid.

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